Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical–fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death.


Introduction
Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and a major public health problem in Europe, associated with high mortality and morbidity rates and a significant socioeconomic burden [1][2][3].Worldwide, the annual incidence of head injury is estimated to be between 27 and 69 million new cases, with road traffic accidents being the leading cause [4].
The pathophysiology of TBI is complex and involves a cascade of events that occur at the time of injury and continue for days or weeks after injury [5].In particular, TBI can be attributed to two different, partly overlapping, pathogenetic mechanisms, which can be divided into primary and secondary brain damage [6][7][8].Primary brain damage results from direct mechanical damage to brain tissue, which may be caused by acceleration or deceleration forces, rotational forces, or direct impact.The resulting damage may include contusions, lacerations, and diffuse axonal damage [9,10].Secondary brain damage follows the activation of biochemical cascades of various types (such as free radical generation, neuroinflammatory response, and cell death) that occur after the initial injury and may lead to further brain tissue damage, including hypoxia, ischaemia, inflammation, and oxidative stress [11,12].
The study of TBI is of great forensic relevance.The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative [13].Even in the forensic setting, the diagnosis of brain injury may be difficult, as in cases of diffuse axonal injury (DAI) in which macroscopic examination does not reveal obvious brain changes [14,15].
Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time [16].They may also represent valuable therapeutic targets for the treatment of TBI [17][18][19][20][21].
In this sense, micro-RNAs (miRNAs) represent potential and promising biomarkers applicable in clinical diagnostics and forensic science.miRNAs are small non-coding RNA molecules involved in the pathophysiology of many diseases and are crucial for neurodevelopment and brain function [22,23].By regulating gene activity, miRNAs control cellular processes for neuronal damage and repair: differentiation, proliferation, apoptosis, and metabolism.Altered miRNA levels have been reported in several central nervous system (CNS) disease processes [24], including traumatic brain injury [25].miRNAs have also been proposed as therapeutic targets for TBI, so several studies have been conducted on murine models in order to evaluate their use [20,21].For example, the use of inhibitors (antagomiR) or mimetics (agomiR) of miRNAs has been tested as an innovative therapeutic strategy to reduce brain damage and improve clinical outcomes in patients with TBI [26,27].
Compared to known TBI protein biomarkers, miRNAs may be preferable due to their specific characteristics [28].First, their small size is related to greater stability even in highly degraded samples, such as cadaveric samples.Secondly, their high tissue-specific expression gives them greater sensitivity to the pathology under investigation.Furthermore, due to their action at the post-transcriptional level, miRNAs can be detected in the early stages of a disease, long before the effects of downstream protein expression are observed [29].For these reasons, several papers have been produced to evaluate the different miRNA expression profiles in subjects with TBI compared to controls [30].
The majority of TBI studies have been conducted on cell lines or biological fluid samples such as plasma, serum, cerebrospinal fluid (CSF), and saliva [31][32][33][34][35].Only a minority of studies have been conducted on tissue samples (brain tissue), mainly from mouse models subjected to controlled cortical impact [36][37][38][39].In the currently available postmortem studies of TBI, the expression levels of specific miRNAs on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples have been investigated.In these studies, several miRNAs have been identified as dysregulated in brain injury, such as miR-16, miR-21, miR-92, miR-124, miR-138, and miR-144 [40,41].Numerous other miRNAs have been implicated in the pathophysiology of TBI and are being investigated for their potential role in both clinical-therapeutic and forensic settings.Despite this evidence, further studies are currently required to validate their use.
This study concerns the profile expression of a set of miRNAs, preliminarily selected based on the literature review, evaluated on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples from subjects who died of TBI, stratified based on the survival time and the severity of the injury.
The aim of this study is to investigate the expression profiles (downregulation and upregulation) of a set of miRNAs in subjects deceased as a result of TBI in order to assess, verify, and define the role of non-coding RNA molecules in different pathophysiological mechanisms of brain injury.This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma.

Study Group Characteristics
Forty subjects deceased as a result of traumatic brain injury (TBI) were selected: thirtyone (77.50%) were male and nine (22.50%) were female.The mean age of the subjects included in the study group was 45.05 ± 19.17 SD (median 53 years, age min 12, max 65).In particular, 13 subjects were younger than 35 years old, and 27 were older than 35 years old.
The most common cause of death in the study group was major trauma (55%).The injury mechanisms leading to the head injury were a road traffic accident in twenty-five cases (62.5%), a fall from standing in seven cases (17.5%), a fall from a height in four cases (10%), a train accident in three cases (7.5%), and blunt trauma in one case (2.5%).Road traffic accidents were thus the most common mechanism of head injury, accounting for 42.5% of the study group.The most common type of brain injury was subarachnoid haemorrhage (SAH), which was present in 24 of the subjects (60%).In all selected cases, the postmortem interval (PMI) was less than 6 days, with a mean of 80.275 ± 19.599 h (median 76, PMI min 52, PMI max 123).In addition, of the selected subjects, six had undergone surgical treatment by decompressive craniotomy (Table 1

Control Group Characteristics
Ten subjects who died of natural causes, with a cardiovascular genesis, were selected according to the inclusion and exclusion criteria (Table 2).Six (60%) were male and four (40%) were female, and the mean age of the subjects included in the study group was 53.2 ± 10.97 SD (median 55.5 years, age min 24, max 65).

Subgroup Characteristics
The study group was selected by defining four subgroups consisting of cases distributed according to survival time (Figure 1).Specifically, the four subgroups consisted of twelve cases of fatal TBI (no survival), nine cases of TBI with short survival (survival < 1 day), ten cases of TBI with middle survival (survival >1 and <7 days), and nine cases of TBI with long survival (survival > 7 days).The study group was stratified in parallel according to the severity of the trauma, which was assessed using criteria derived from the Abbreviated Injury Scale (AIS) score (Figure 2).Specifically, three subgroups were defined, consisting of eight cases of mildmoderate TBI (AIS 1-2), ten cases of severe TBI (AIS 3-4), and twenty-two cases of criticalfatal TBI (AIS 5-6).
In contrast, no significant variations in expression levels between cases and controls were detected for miR-19a-3p.

Subgroup Analysis: Correlation of Expression Profiles with Survival Time and Severity of Trauma
According to survival time, data analysis showed that miR-21-5p expression levels were approximately three times higher in the subgroup consisting of TBI with short survival (<1 day) than in the subgroups with no survival (fatal) and middle survival (1-7 days).In contrast, miR-21-5p expression levels were under-regulated in the subgroup of TBI with long survival (>7 days).
The analysis of miR-16-5p showed an approximately 6-fold upregulation in the shortsurvival subgroup (<1 day) and an approximately 4-fold upregulation in the middle-sur- In contrast, no significant variations in expression levels between cases and controls were detected for miR-19a-3p.

Subgroup Analysis: Correlation of Expression Profiles with Survival Time and Severity of Trauma
According to survival time, data analysis showed that miR-21-5p expression levels were approximately three times higher in the subgroup consisting of TBI with short survival (<1 day) than in the subgroups with no survival (fatal) and middle survival (1-7 days).In contrast, miR-21-5p expression levels were under-regulated in the subgroup of TBI with long survival (>7 days).
The expression levels of miR-130a were significantly higher in TBI cases with short (<1 day) and middle survival (1-7 days) than in TBI cases with no survival (fatal) or long survival (>7 days).
In contrast, there were no statistically significant differences in the expression levels of the remaining miRNAs investigated between the subgroups stratified by survival time.
In relation to the severity of injury, data analysis showed a significantly higher expression of miR-16-5p and miR-21-5p in the critical-fatal TBI subgroup (AIS 5-6) than in the mild-to-moderate TBI (AIS 1-2) and severe TBI (AIS 3-4) subgroups.In contrast, there was no statistically significant variation in the expression levels of the remaining miRNAs investigated between the subgroups stratified by TBI severity (Figure 4).was no statistically significant variation in the expression levels of the remaining miRNAs investigated between the subgroups stratified by TBI severity (Figure 4).

Discussion
The mechanisms of injury described after TBI include neuroinflammation, synaptic dysfunction, protein aggregation, oxidative stress, blood-brain barrier damage, cerebral oedema, and cell death [42].There are several cell populations involved in the pathophysiology of TBI, including astrocytes, microglia, oligodendrocytes, and endothelial cells, with inflammatory and reactive responses that may contribute to brain damage [43,44].After TBI, cells of the innate immune system, such as microglia, can rapidly respond to the traumatic event by releasing cytokines and other pro-inflammatory molecules that contribute to the inflammatory response and local brain inflammation [45][46][47].Cells of the

Discussion
The mechanisms of injury described after TBI include neuroinflammation, synaptic dysfunction, protein aggregation, oxidative stress, blood-brain barrier damage, cerebral oedema, and cell death [42].There are several cell populations involved in the pathophysiology of TBI, including astrocytes, microglia, oligodendrocytes, and endothelial cells, with inflammatory and reactive responses that may contribute to brain damage [43,44].After TBI, cells of the innate immune system, such as microglia, can rapidly respond to the traumatic event by releasing cytokines and other pro-inflammatory molecules that contribute to the inflammatory response and local brain inflammation [45][46][47].Cells of the adaptive immune system, such as T lymphocytes and B lymphocytes, may also be involved in the response to TBI through a more specific response that can be activated after significant tissue damage [48,49].
miRNAs represent potential and promising biomarkers of TBI, and several studies have evaluated their expressivity and demonstrated their dysregulation in relation to TBI, suggesting their involvement in pathophysiological mechanisms and their possible use in defining the diagnosis and prognosis of TBI [50,51].
miRNAs act through the mechanism of gene expression regulation by binding to specific target genes, called "target mRNAs", and regulating their expression at the transcriptional or translational level [52,53].This affects signalling pathways and cellular processes involving specific target genes regulated by different miRNAs.
The present study identified a panel of miRNAs that may be related to the mechanisms of damage in different brain cell populations, as well as those associated with neuroprotective and neurodegenerative mechanisms.The expression of these miRNAs was evaluated in brain tissue sections taken from subjects deceased as a result of TBI.This was compared with the expression of the same miRNAs in brain tissue sections taken from a control group of subjects deceased as a result of natural causes of cardiovascular origin.
The described role of miR-16 [42,43,54,55] in relation to TBI is complex, depending on the specific context and conditions studied.miR-16 influences specific signalling pathways involved in traumatic brain damage and has been particularly linked to the regulation of several target genes involved in cell proliferation and glial apoptosis, including CDK6 (Cyclin-Dependent Kinase) and BCL-2 (B-cell lymphoma 2).Specifically, miR-16-5p overexpression can induce apoptosis by directly regulating BCL-2, an anti-apoptotic protein that prolongs cell survival by neutralising pro-apoptotic factors.The aberrant downregulation of BCL-2 disrupts mitochondrial membrane integrity, induces the mitochondrial release of pro-apoptotic proteins (e.g., cytochrome C and AIF), and triggers caspase activation and cytoskeletal degradation, leading to apoptosis.Studies conducted on glioma cell lines have shown that miR-16-5p binds directly to the 3 ′ UTR of BCL-2 mRNA and deregulates cellular levels of BCL-2 mRNA and protein.These studies support an inhibitory role of miR-16-5p on glial cell proliferation, as well as its stimulating effects on apoptosis and increased caspase activity.MiR-16 has also been studied in relation to other brain disorders, such as Alzheimer's disease, which may share some pathological features with TBI.It has been linked to the deposition of β-amyloid (Aβ), whose neurotoxicity is known to be a major cause of neurodegeneration.Additionally, miR-16 has been found to be significantly dysregulated in TBI patients, with significantly higher expression levels, particularly within the first 24 h post-trauma.
MiR-21 [38,[46][47][48], one of the most studied miRNAs, is known to be involved in various cellular processes, including inflammation, apoptosis, and cell proliferation, and plays an important role in various pathological conditions, including traumatic brain injuries.In particular, in TBI, miR-21 expression is involved in blood-brain barrier damage, apoptosis, cell proliferation and differentiation, autophagy processes, and oxidative stress.Studies have shown that miR-21 acts at the astrocyte population level (reducing their activation), at the macrophage level (with pro-inflammatory effects by acting on the STAT3 signalling pathway, a protein involved in regulating the inflammatory response and cell activation), on dendritic cells, and on the T-cell line.In particular, miR-21 has been implicated in the regulation of the PTEN/PI3K/AKT pathway, affecting the expression of PTEN, which acts as an inhibitor of the PI3K/AKT pathway involved in cell survival and proliferation.MiR-21 can also influence the regulation of genes and proteins involved in blood-brain barrier permeability, indirectly affecting the inflammatory response and repair capacity.Recent studies have demonstrated high levels of miR-21 expression after TBI, and treatment with antago-miR-21 has been proposed as a potential therapy to reduce blood-brain barrier (BBB) damage [56,57].A particular over-regulation of miR-21 has also been observed in the serum of patients with severe TBI (sTBI) compared to those with mild TBI (mTBI), at very early times and up to 15 days after the trauma.Furthermore, no increase was found in patients with musculoskeletal injuries without TBI, which is why miR-21 has been considered a potential new biomarker for TBI and a future therapeutic target [58].
MiR-23a [49] has been found to play a suppressive role in neuronal apoptosis and the inflammatory response in murine models of TBI.PTEN has been identified as a potential target gene of miR-23a.Specifically, studies have shown that miR-23a negatively regulates the expression levels of PTEN mRNA in primary cortical neurons, suppressing its expression both in vitro and in vivo.Since PTEN is a negative regulator of the AKT/mTOR pathway, it has been hypothesized that miR-23a plays a protective role in TBI by acting on the PTEN/AKT/mTOR pathway.Studies have shown reduced expression levels of miR-23a in TBI cases compared to controls, and the potential role of agomiR-23a as a therapeutic target has been evaluated.
MiR-130a [50,51], a micro-RNA involved in cellular and pathological processes, appears to play an important role in the context of traumatic brain injury (TBI).Recent studies have suggested that miR-130a may influence various aspects of the brain response to traumatic damage.Some of the target genes of this miRNA in the context of TBI include those involved in the regulation of inflammation, such as tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), as well as genes that regulate cell proliferation and cell death.miR-130a can influence the gene expression of PTEN, Homeobox Hox-A5, and AQP4 (aquaporin 4), leading to apoptosis, BBB damage, and brain oedema.A study conducted to investigate the role of miR-130a in BBB damage found an increase in its expression levels in microvascular endothelial cells and a corresponding increase in BBB permeability and brain oedema.It has been shown that the use of Antagomir-130a, an antagonist of miR-130a, could attenuate brain oedema, reduce BBB permeability, reduce brain lesion volume, and improve neurological function.
MiR-155 [52,53] has been implicated in the regulation of macrophage signalling mediated by Toll-like receptors (TLRs) 3 and 4, as well as interferon (IFN)-γ.This miRNA plays a key role in the modulation of neurotoxicity, macrophage and microglial activation, and in the regulation of the release of inflammatory mediators such as nitric oxide, cytokines, interleukins, and chemokine signalling.Recent studies have shown that experimentally induced traumatic brain injury (TBI) increases miR-155 expression in the damaged cortex and hippocampus [59,60].Additionally, these injuries induce neuroinflammatory responses mediated by microglial cells, which are associated with neuronal loss and persistent neurological deficits [61,62].Given the documented pro-inflammatory role of miR-155 in neurodegenerative diseases, its alteration in microglia induced by TBI may contribute to the chronicity of neuroinflammation and related neurodegeneration.
Therefore, according to the analysis of the data obtained in the present study, it can be argued that the upregulation of miR-16-5p, miR-21-5p, miR-130a-3p, and miR-155-5p confirms the role of these miRNAs with pro-inflammatory, pro-apoptotic, and regulatory effects in signalling pathways involved in cell permeability and blood-brain barrier damage (Figure 5).
The downregulation of miR-23a-3p was consistent with the literature studies highlighting a potential protective role of this miRNA in TBI, confirming its suppressive effect on neuronal cell apoptosis and the inflammatory response (Figure 5).
Regarding the survival time associated with TBI, the significantly higher expression levels of miR-21-5p in the short-survival subgroup compared to the fatal TBI and middlesurvival subgroups suggest that miR-21-5p may be associated with a rapid and specific response to severe injury.The approximately six-fold increase in miR-16-5p expression levels in the short-survival group and the approximately four-fold increase in the middle-and long-survival subgroups may indicate a key role of miR-16-5p in the response to TBI and short-term survival.Finally, the significant increase in miR-130a expression levels in TBI cases with short and middle survival suggests that miR-130a may be involved in the initial pathophysiological response following trauma.
were found between the study group and the control group for miR-19a-3p (Figure 3).
Therefore, according to the analysis of the data obtained in the present study, it can be argued that the upregulation of miR-16-5p, miR-21-5p, miR-130a-3p, and miR-155-5p confirms the role of these miRNAs with pro-inflammatory, pro-apoptotic, and regulatory effects in signalling pathways involved in cell permeability and blood-brain barrier damage (Figure 5).
The downregulation of miR-23a-3p was consistent with the literature studies highlighting a potential protective role of this miRNA in TBI, confirming its suppressive effect on neuronal cell apoptosis and the inflammatory response (Figure 5).In relation to trauma severity, the significant increase in miR-16-5p and miR-21-5p expression levels in the fatal-critical TBI subgroups (AIS 5-6) compared to the mild-moderate TBI (AIS 1-2) and severe TBI (AIS 3-4) subgroups suggests that these miRNAs may be involved in specific responses to severe injuries and trauma severity.
Overall, the results obtained from the data analysis suggest that miR-21-5p, miR-16-5p, and miR-130a may play an important role in the response to TBI and in determining the survival time and trauma severity in TBI patients.

Selection of Study Population
This multicentre collaborative study was carried out by the University of Salerno, the Sapienza University of Rome, and the University of Foggia.
The study population was selected from the cases that underwent autopsies on the orders of the Judicial Authority at the Forensic Medicine Departments, in a period between 2019 and 2023.In particular, in all the selected autopsy cases, tissue samples were taken and subsequently fixed in formalin and embedded in paraffin (FFPE) for subsequent histo-pathological examination.
A study population consisting of 40 cases of subjects with TBI-related deaths (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group) was then selected.

Sample Collection
For each case, a sample of brain tissue taken at autopsy was examined, fixed in formalin, and then embedded in paraffin (FFPE).
A total of 50 formalin-fixed, paraffin-embedded (FFPE) brain tissue samples were then processed, of which 40 belonged to the study group (corresponding for each case to the area of brain tissue most affected by the traumatic injury, selected on the basis of the findings of both macroscopic and microscopic autopsy examinations performed in all cases selected for this study) and 10 belonged to the control group.

Total RNA Extraction
Four 10 µm thick sections were obtained from each formalin-fixed, paraffin-embedded (FFPE) tissue sample, and total RNA was isolated using the miRNeasy FFPE kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.
Briefly, the protocol included an initial deparaffinization phase in which 1 mL of xylene and then 1 mL of ethanol were added to the tissue sections.The sample was digested with 240 µL PKD buffer and 10 µL of Proteinase K, incubated at 56 • C for 15 min and then at 80 • C for 15 min.It was then treated with DNase Booster Buffer in an amount equal to one-tenth of the total sample volume (approximately 16 µL) and with 10 µL of DNase stock solution and incubated at room temperature for 15 min.The sample was then treated with 320 µL RBC buffer, and 1120 µL of ethanol was added to the total lysate.Finally, the entire sample was transferred in two steps through the RNeasy MinElute spin column and centrifuged for 15 s at ≥8000× g (≥10,000 rpm).Total RNA was eluted in 17 µL of nuclease-free water.The RNA concentration was measured using a Nanodrop spectrophotometer with 1 µL of isolated RNA.Samples were stored at −80 • C.

Reverse Transcription and Real-Time PCR Assay of miRNA
For miRNA detection, 10 ng of total RNA, assessed using NanoDrop spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA), was processed using cDNA Synthesis Kit for reverse transcription (RT) of the miRNAs.
Real-time PCR (RT-qPCR) was performed for each miRNA (Table 7), using Taq DNA Polymerase and miRNA Assays Kits.Reverse-transcribed cDNA was diluted at a 1:10 ratio, and a total reaction volume of 20 µL was used and incubated at 95 • C for 20 seconds, 95 • C for 3 s (for 40 cycles), and 60 • C for 30 s (for 40 cycles).Each reaction was performed in duplicate.For relative quantification, the 2-∆Ct method was applied, using the geometric means of miRNA hsa-miR-92a-3p as housekeeping.

Statistical Analysis
To achieve reproducibility and statistical significance, analyses were performed in duplicate for cases and controls.For normally distributed variables, a t-test or ANOVA was used.The association of the relationship between the miRNAs studied and the categorical variables was analysed using the Kruskal-Wallis test.A p-value of p < 0.05 was considered statistically significant.The statistical programme IBM SPSS STATISTICS was used for data analysis.

Conclusions
The results of this study provide significant evidence for the potential of the miRNAs studied as highly predictive biomarkers in distinguishing TBI cases from controls.These miRNAs could open up new perspectives for the postmortem diagnosis of TBI, offering the possibility of determining survival and severity.
Furthermore, the differential expression of the miRNAs studied according to trauma severity and survival time suggests that these miRNAs may also be valuable therapeutic targets.Indeed, since miRNAs are crucial regulators of many biological processes, including inflammation, apoptosis, and tissue repair, they could be modulated to develop new therapeutic strategies.In this sense, several studies in mouse models have already highlighted the potential role of miRNAs as therapeutic targets in TBI.However, the translation of basic research findings into clinical practice will require further studies, particularly to better understand the safety and efficacy of miRNA modulation in vivo.
In conclusion, the significant predictive power of these miRNAs could revolutionise the approach to the postmortem diagnosis of TBI, greatly improving the ability to identify and assess such injuries.These results suggest that miRNA analysis could become an essential tool in forensic investigations in the future, providing more accurate and detailed information about the nature and extent of TBI.The predictive power in relation to the timing and severity of trauma could ultimately contribute to the definition of the circumstances of death.

Figure 1 .
Figure 1.Distribution of study group by survival time expressed in days.

Figure 1 .
Figure 1.Distribution of study group by survival time expressed in days.

Figure 1 .
Figure 1.Distribution of study group by survival time expressed in days.

Figure 2 .
Figure 2. Distribution of study group by severity of trauma, as defined by AIS score parameters.

Figure 2 .
Figure 2. Distribution of study group by severity of trauma, as defined by AIS score parameters.
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Figure 4 .
Figure 4. Clustered heat map showing the differential expression of the 5 miRNAs in the 3 subgroups defined on the basis of survival time (left figure) and on the basis of trauma severity (right figure).The coloured boxes represent the relative expression of the group (measured by Pearson's distance metric), and the miRNAs are clustered in the heat map using a comprehensive clustering algorithm.

Figure 4 .
Figure 4. Clustered heat map showing the differential expression of the 5 miRNAs in the 3 subgroups defined on the basis of survival time (left figure) and on the basis of trauma severity (right figure).The coloured boxes represent the relative expression of the group (measured by Pearson's distance metric), and the miRNAs are clustered in the heat map using a comprehensive clustering algorithm.

Figure 5 .
Figure 5. Expressivity (up-and downregulation) of the miRNAs examined in this study and their correlation with pathways, pathophysiological effects, and target cell populations.

Figure 5 .
Figure 5. Expressivity (up-and downregulation) of the miRNAs examined in this study and their correlation with pathways, pathophysiological effects, and target cell populations.

Table 2 .
Control group characteristics.

Table 5 .
Definition of subgroups based on severity of trauma.death, partly or fully damaged brainstem or upper part of cervical area due to pressure or disruption.Fracture and/or wrench of upper part of cervical area with injuries of spinal cord.

Table 6 .
Characteristics of the miRNAs selected for this study.

Table 7 .
Micro-RNA sequences used in PCR.